Panels depict biochemical redox cycles of (A) single Cys and (B) disulfide-forming pair of Cys residues and their evolution (white central vertical panel). Incorporation of a single Cys (α) may make the protein immediately responsive to a range of oxidative modifications, including nitrosation and glutathionylation. Cys with lower pKas are more susceptible to modifications. Introduction of a second Cys (β) at a later stage may then enable disulfide formation, preventing overoxidation of the low pKA “active” Cys and effectively forming a bistable redox switch. Because Grxs are homologs to Trxs and share similar binding modes of the protein substrate, the complete redox cycle may potentially be immediately effected. These evolutionary modifications are reversible i.e., the resolving Cys may be lost, returning the protein to the Grx pathway etc.

Evolution of CD4 (A) phylogenetic tree in mammals. The color of the branches indicates the number of Cys of the CSD motif in D2. The CSD is found in species with red branches. Species with purple branches have only the C-terminal Cys. The CSD was likely acquired in primates (A–D) around 75 MYA, and independently in muroid rodents viz mice, rats (O) and hamsters (P), after the divergence of squirrels (Q, sciuridae), also around 75 MYA. Acquisition of the CSD in muroids was accompanied by rapid evolution of the CD4 protein as evidenced by the long branch lengths of this clade (upper red branches). The four major clades of Eutherian mammals are Afrotheria (including elephants E); Xenartha (none depicted); Laurasiatheria (F–L, shown in pink) and Euarchontaglires (primates, A–D, green circle; glires, N–S yellow circle; and treeshrews, M) (Murphy et al., ) Key: A, Lesser apes; B, Greater apes; C, Marmoset (Platyrhines); D, Galago; E, African Elephant; F, Bats; G, Panda/Mink/Ferret; H, Dog and cat; I, Pig; J, Dolphin, Whale and Camel; K, Sheep, Bovine, Goat; L, Horse; M, Tree shrew; N, Rabbit; O, Mouse and rat; P, Chinese hamster; Q, Squirrel/wood chuck; R, Guinea pig; S, Mole rat; T, Wallaby and opossum; U, Platypus. (B) Evolution of CD4 and related molecules showing important thiol-containing sites. Molecules are depicted from the more ancestral forms of the molecule (leftmost) to more recently evolved forms to the right. The CD4 homologs consist of two to four extracellular Ig domains, a single transmembrane region and a short cytoplasmic tail. Three important thiol-bearing functional sites have been identified: the CXC motif in the cytoplasmic tail, the between-sheet disulfide in D1, and the cross-strand disulfide (CSD) in D2. The CXC motif is present in all CD4 orthologs (β–ε), but not in the CD4-like LAG3 molecule. The between-sheet disulfide in D1 is present in CD4 and LAG3 (α–ζ). In some teleosts, such as trout and fugu, the Cys on strand B is lost, leaving a single unpaired Cys on strand F. D1 can be definitively identified in CD4REL because it is distinctively encoded on two exons in molecules β–ε (Laing et al., ). It is involved in interaction with MHC II molecules. Thiols in the D2 site have evolved over time. Originally D2 contained Cys residues on strands B and F, typical of Ig domains (α–γ). Around 250 MYA, the thiol on strand B was lost (δ–ε). Around 75 MYA, a new thiol appeared on strand C allowing the novel CSD to form upon oxidation (ζ). α–γ are CD4-like proteins which evolved in a trout ancestor around 450 MYA. Interacting proteins are shown in gray: all proteins bind MHC class II; the CD4-related molecule LAG3 (α) binds MHC but not LCK. D3 and D4 contain glycosylation sites which may serve to fend off adventitious interactions with other membrane proteins (Laing et al., ). Ig domains are depicted as bisected hexagons with each half representing one sheet: V-like domains are depicted in green; C-like domains in magenta. A CXXC motif in LCK binds to the CXC motif of CD4 via Zn²⁺ (depicted as a blue diamond) (Lin et al., ). The extracellular and cytoplasmic sides of the membrane are labeled on the right.

Multiple sequence alignment of CD4 in the region of the CSD in species where both Cys are conserved (upper panel) and measures of its conservation (lower panel) across two groups: primates and muroids (purple), and primates only (green). For each group, the conservation of the amino acid residue in each column, quality (BLOSUM62 score based on observed substitutions) and consensus (most common residue and its proportion for each column) as determined by Jalview (Waterhouse et al., ) is shown. The sequence alignment of CD4 showed less conservation in the vicinity of the CSD motif. In the portion depicted, the average conservation across the primate + muroid lineages is 6.2 for residues 53–80 (CSD region highlighted in yellow) and 7.9 within the primate group.

(A) Evolution of the ERO1 gene family. Branches are colored according to the number of Cys present in the NTR of each species, as shown in the legend. A single ERO1-like ancestral gene (pink background) is present in eukaryotes from yeast to lamprey (J–P). With the exception of plants (P), the majority of these genes have four Cys in the NTR (red branches). ERO1 duplicated prior to the divergence of bony fishes. ERO1A (green background) genes retained the nested Cys motif, as does ERO1B (yellow background) in early diverging species such as fish, sharks, and amphibians (F, G). However, later diverging species such as birds, reptiles, and mammals (H, I) have only two Cys, homologs to the inner pair of CSD-forming Cys. Plants (P) have an additional Cys, C-terminal to the nested Cys motif. Selective loss of Cys residues has resulted in a single N-terminal Cys in some ERO1 genes (purple branches) such as in ERO1L in tapeworm (O) and Silk moth (K), and ERO1B in the Japanese ricefish. Key to branches: A, Mammals; B, Birds; C, Frog and turtle; D, Shark and Spotted gar; E, Fish; F, Fish; G, Shark, Frog and Coelacanth; H, Birds/Snake/Chameleon; I, Mammals; J, Sea squirt/Oyster/Sea urchin; K, Flies (Mosquito/Beetle/Bugs); L, Ants; M, Lancelet and Polychaete worm; N, Worms; O, Tapeworms; P, Plants. (B) Line diagram of ERO1 showing the disulfides and NTR. The two CSDs are the two nested disulfides in the NTR region. The NTR region comprising residues 1–55 is typically deleted in yeast functional assays. An equivalent region is typically deleted in ERO1α (Hansen et al., ). Interestingly, this region is not generally deleted in ERO1B (Pagani et al., ).

Evolution of AKT genes showing the presence of the CSD. Colors of branches denote the number of Cys residues homologous to those of the CSD present in each species. Reversion to a single C-terminal Cys in the Chinese tree shrew is accompanied by rapid diversion of the sequence from other mammalian sequences. Keys to branches A, Mammals, birds, reptiles; B, Fishes; C, Sea lamprey; D, Mammals; E, Snakes; F, Spotted gar, salmon, and fishes; G, Frogs; H, Elephant fish; I, Zebra fish; J, Mammals; K, Tasmanian devil, Opposum, Turtle, and Birds; L, Frog and Coelacanth; M, Fishes; N, Acorn worm; O, Sea squirt; P, Trichina worm; Q, Eye worm, Pig round worm, and Pinewood nematode worm; R-C. elegans, pole worm and nematode worm; S-Y-Arthropods including Lepidoptera (T) and Diptera (U,V).

Location of CSDs in the secondary structure of the proteins studied according to PDB structures: CD4—4h8w, ERO1—3ahq, and AKT—1unr. The Cys on the orange strand is the putative active Cys for each protein. Trx/Grx must bind as an extra strand near the active Cys as shown for AKT. In ERO1 and CD4, this site is protected by an additional strand (strand G in both CD4 and ERO1), which must be displaced to allow access by Trx. For CD4 to become accessible to Trx/Grx, one of the hairpins must hinge open to allow Trx/Grx to dock in place of the strand as shown in the hypothetical CD4^* diagram.